S-Adenosylhomocysteine hydrolase is the only enzyme in mammalian cells for the removal of adenosylhomocysteine, the end-product of biological transmethylation reactions. For this reason, the enzyme is critical for the regulation of adenosylmethionine-dependent methylations. We have used several approaches to investigate structure/function relationships of AdoHcy hydrolase. We have cloned and expressed the cDNA for the enzymes from rat liver, D. discoideum, and Rhodobacter capsulatus. The amino acid sequences are highly conserved between species suggesting that much of the sequence is required for enzyme function. Human and bacterial amino acid sequences have 64% sequence identity; to our knowledge this is the highest level of conservation reported between human and prokaryotic enzymes. The human and rat sequences differ from that of Rhodobacter by an additional segment of 36 amino acids that is present in the Rhodobacter sequence. Site- directed mutagenesis and protein modification studies of rat AdoHcy hydrolase have allowed us to assign amino acids 213 to 244 a role in the binding of NAD, and Cys78, Cys1l2 and Cys52 a location near the AdoHcy binding site. These studies have also indicated that amino acids 1 to 112 are less tightly folded than the remainder of the hydrolase molecule. Mammalian AdoHcy hydrolase is expressed at a significantly higher level in liver compared to other tissues, indicating transcriptional control of expression. To study the transcriptional control of hydrolase expression, the gene for rat AdoHcy hydrolase has been cloned. The gene consists of 10 exons with GC boxes preceding the transcription start.